In immature bone marrow derived dendritic cells (BMDC) caspase 3 acting in a non-apoptotic pathway controls the processing of molecules required for endosomal remodeling into tubular structures. This activity allows delivery of proteins involved in antigen presentation to the cell membrane. The importance of caspase 3 in this process has been previously analyzed using caspase inhibitors. In this application it will be further characterized by analyzing DC from caspase 3-/- mice or after "in vitro" silencing of caspase 3 in immature BMDC using Lentiviral vectors. "In vivo" experiments are also proposed where caspase 3 will be silenced in CD11c positive cells only and the impact on the immune response analyzed. Levels of active caspase 3 in immature BMDC are very low (5-8% of what observed during apoptosis). The hypothesis that the low amount of caspase 3 activity is selectively contained at sub-cellular locations will also be determined using confocal and electron microscopy as well as sub-cellular fractionation. "In situ" trapping will be performed to pull down the molecular form of active caspase 3 since western blotting indicates that in bone marrow dendritic cells a 29 kDa form of caspase 3 is present in addition to the 32 kDa caspase 3 pro-enzyme. The hypothesis that the 29-kDa form is either functionally active, per se, or more easily cleavable will be evaluated using in situ trapping and functional overexpression. Finally the biochemical characterization of caspase 3 activity on target proteins (AP-1 and dynamin) will be evaluated using gel filtration, site-directed mutagenesis and overexpression experiments with the ultimate goal of understanding how truncated or full length proteins are functionally involved on endosomal remodeling in maturing BMDC. Mature dendritic cells are the most powerfull initiator of an immune response. Thus any molecule interfering with the maturation process will have a profound impact on their immunological function. We previously found that low levels of caspase 3 activity is important in maintaining dendritic cells in an immature state. The major focus of this proposal will be to further our knowledge on caspase 3 control of dendritic cell maturative processes.